Antibiotic resistance in Gram-negative bacteria, in general, and Escherichia coli (E. coli), the leading cause of urinary tract and bloodstream infections, in specific, is a common occurrence; however, the development of new antibiotics to treat these bacteria is hindered by the strong permeability barrier posed by the outer membrane (OM). Understanding changes that occur to this barrier in differing environmental conditions will inform antibiotic discovery strategies that lead to new clinically relevant antibiotics. The objectve of this proposal is to elucidate changes occurring to the E. coli OM during nutrient limiting conditions that may affect the cell's resistance to harsh conditions with the long-term goal of understanding bacterial stress responses and the ways they can be manipulated. OM permeability to anionic detergents such as SDS serves as a good model for OM antibiotic permeability in cells will low metabolic activity. Preliminary data demonstrate that exponentially growing cells, carbon-limited cells, and nitrogen-limited cells are all resistant to high concentrations of SDS; however, in exponential phase, this resistance is based on the presence of efflux pumps suggesting that the SDS crosses the OM while, in stationary phase, the resistance is independent of efflux suggesting the membrane is strengthen to decrease its permeability. Furthermore, the stationary phase resistance can occur through at least two pathways, an rpoS-dependent pathway in carbon-limited conditions and an rpoS-independent pathway in nitrogen-limited conditions. Neither of these pathways involves the non- essential envelope stress responses nor the nitrogen starvation regulators, rpoN and ntrC, suggesting that the pathways are highly novel. Therefore, this proposal will address through Aims with the follow goals the hypothesis that changes occur to the OM during stationary phase making the OM more resistant to detergents and that both rpoS-dependent and rpoS-independent pathways can induce these changes. Specific Aim 1: Determine if changes in OM physiology occur between exponential, C-limited stationary, and N-limited stationary phase Specific Aim 2: Determine what rpoS-dependent and rpoS-independent pathways lead to decreased SDS permeability during stationary phase Through completion of these Aims, any changes to OM lipopolysaccharide modification, phospholipid levels or localization, and/or OM protein expression or levels will be elucidated. In addition, through transposon mutagenesis and Tn-Seq approaches, the pathways that lead to decreased OM SDS permeability in stationary phase will be determined. Understanding of the OM changes that are occurring in nutrient limiting conditions may lead to improvements in antibiotic discovery strategies that identify antibiotics effective for Gram-negative bacteria in varying environmental conditions.